Fungal Genome Sequences Research Articles

An investigation into the transcription factors and regulatory mechanisms of Aspergillus

AbstractAflatoxins produced by Aspergillus flavus, a group of filamentous fungi widely distributed in nature as foodborne pathogens, are a major threat to food safety. It is thus clear that Aspergillus is inextricably linked to the food sector. Transcription factors enable the correct expression of genes at specific spatial and temporal levels, and they constitute a considerable proportion of the fungal genome sequence. Their presence is particularly important for growth, secondary metabolism, stress response, and pathogenic process of Aspergillus. This article reviews the functional regulatory mechanisms of important transcription factors in the foodborne pathogen Aspergillus flavus and other Aspergillus species, the current status of research technologies, and the potential value of applying them to diverse fields such as food safety, biotechnology, medical health, and agricultural output in the future.

Global Analysis of Natural Products Biosynthetic Diversity Encoded in Fungal Genomes.

Fungal secondary metabolites (SMs) represent an invaluable source of therapeutic drugs. Genomics-based approaches to SM discovery have revealed a vast and largely untapped biosynthetic potential within fungal genomes. Here, we used the publicly available fungal genome sequences from the NCBI public database, as well as tools such as antiSMASH, BIG-SLiCE, etc., to analyze a total of 11,598 fungal genomes, identifying 293,926 biosynthetic gene clusters (BGCs), which were subsequently categorized into 26,825 gene cluster families (GCFs). It was discovered that only a tiny fraction, less than 1%, of these GCFs could be mapped to known natural products (NPs). Some GCFs that only contain a single BGC internally are crucial for the biodiversity of fungal biosynthesis. Evident patterns emerged from our analysis, revealing popular taxa as prominent sources of both actual and potential biosynthetic diversity. Our study also suggests that the genus rank distribution of GCF is generally consistent with NP diversity. It is noteworthy that genera Xylaria, Hypoxylon, Colletotrichum, Diaporthe, Nemania, and Calonectria appear to possess a higher potential for SM synthesis. In addition, 7213 BGCs match possible known compound structures, and homologous gene clusters of well-known drugs can be located in different genera, facilitating the development of derivatives that share structural similarity to these drugs and may potentially possess similar biological activity. Our study demonstrated the various types of fungi with mining potential, assisting researchers in prioritizing their research efforts and avoiding duplicate mining of known resources to further explore fungal NP producers.

Signatures of Mollicutes-related endobacteria in publicly available Mucoromycota genomes.

Mucoromycota fungi and their Mollicutes-related endobacteria (MRE) are an ideal system for studying bacterial-fungal interactions and evolution due to the long-term and intimate nature of their interactions. However, methods for detecting MRE face specific challenges due to the poor representation of MRE in sequencing databases coupled with the high sequence divergence of their genomes, making traditional similarity searches unreliable. This has precluded estimations on the diversity of MRE associated with Mucoromycota. To determine the prevalence of previously undetected MRE in fungal genome sequences, we scanned 389 Mucoromycota genome assemblies available from the National Center for Biotechnology Information for the presence of MRE sequences using publicly available tools to map contigs from fungal assemblies to publicly available MRE genomes. We demonstrate a higher diversity of MRE genomes than previously described in Mucoromycota and a lack of cophylogeny between MRE and the majority of their fungal hosts. This supports the late invasion hypothesis regarding MRE acquisition across most of the examined fungal families. In contrast with other Mucoromycota lineages, MRE from the Gigasporaceae displayed some degree of cophylogeny with their hosts, which may indicate that horizontal transmission is restricted between members of this family or that transmission is strictly vertical. These results underscore the need for a refined process to capture sequencing data from potential fungal endosymbionts to discern their evolution and transmission. Screens of fungal genomes for MRE can help improve the quality of fungal genome assemblies while identifying new MRE lineages to further test hypotheses on their origin and evolution.IMPORTANCEMollicutes-related endobacteria (MRE) are obligate intracellular bacteria found within Mucoromycota fungi. Despite their frequent detection, MRE roles in host functioning are still unknown. Comparative genomic investigations can improve our understanding of the impact of MRE on their fungal hosts by identifying similarities and differences in MRE genome evolution. However, MRE genomes have only been assembled from a small fraction of Mucoromycota hosts. Here, we demonstrate that MRE can be present yet undetected in publicly available Mucoromycota genome assemblies. We use these newfound sequences to assess the broader diversity of MRE and their phylogenetic relationships with respect to their hosts. We demonstrate that publicly available tools can be used to extract novel MRE sequences from assembled fungal genomes leading to insights on MRE evolution. This work contributes to a greater understanding of the fungal microbiome, which is crucial to improving knowledge on the dynamics and impacts of fungi in microbial ecosystems.

Assessing several fungal contaminants and their associated mycotoxins in maize cultivated on cornfields of Republic of Moldova

Maize is an important crop for the Republic of Moldova and one of the crops most contaminated with mycotoxins. Maize grain obtained from plants cultivated on Moldavian cornfields in 2021 and 2022 were tested for mycotoxigenic risk using qPCR with primers to several fungal genome sequences engaged in mycotoxin synthesis and ELISA test to screen total aflatoxins, fumonisin B1, zearalenone, deoxynivalenol and T-2 toxin. Except for T-2 toxin, the mycotoxin concentrations were under the limits of detection and did not exceed maximum admissible levels for unprocessed grain. Concentrations of T-2 toxin in grain samples did not correlate significantly with the quantity of toxigenic F. sporotrichioides. All of the analysed grain samples were contaminated with at least one toxigenic fungus, and 20% of the samples were infected with seven different species of toxigenic fungi. Accumulation of fungi in maize kernels was affected significantly by the season, and generally a decrease was observed in fungal frequency and quantity under drought conditions. However, several toxigenic Aspergillus and Fusarium fungi that are able to produce aflatoxins and fumonisins under improper storage conditions were found in the kernels during the whole period of monitoring.

Strategies for Natural Product Discovery by Unlocking Cryptic Biosynthetic Gene Clusters in Fungi

Recent advancements in genome sequencing and bioinformatic analysis of fungal genomes have revealed that fungi possess cryptic or silent biosynthetic gene clusters (BGCs). This discovery suggests that our understanding of the universe of fungal secondary metabolomes is limited. In this review, we summarize recent strategies for activating cryptic BGCs in fungi, identify fungal secondary metabolites, and highlight their biological activities. We also briefly discuss the isolation and purification methods for these compounds. Our focus is on genetics-dependent and genetics-independent approaches for activating cryptic BGCs in fungi. Using the strategies discussed here, cryptic fungal natural products hold significant potential as a source for the discovery of new drug candidates.

Advances in Synthetic Biology of Fungi and Contributions to the Discovery of New Molecules.

The sequencing of fungal genomes is becoming increasingly accessible, with a wealth of data already available. In parallel, the prediction of the putative biosynthetic pathways responsible for the synthesis of potential new natural products is also increasing. The difficulty of translating computational analyses into available compounds is becoming evident, slowing down a process that was thought to be faster with the advent of the genomic era. Advances in gene techniques made it possible to genetically modify a wider range of organisms, including fungi typically considered recalcitrant to DNA manipulation. However, the possibility of screening many gene cluster products for new activities in a high-throughput manner remains unfeasible. Nonetheless, some updates on the synthetic biology of fungi could provide interesting insights that could help to achieve this goal in the future.

Identifying Fungal Secondary Metabolites and Their Role in Plant Pathogenesis.

Pathogenic fungi are the main infectious agents of plants. Secondary metabolites produced by these fungi, also recognized as natural products, are key mediators of plant-fungal interactions. Knowledge on the biosynthesis of these metabolites, the accessibility to fungal genome sequences, and the development of gene disruption techniques open up opportunities to identify many more of these metabolites both in vitro and in planta. This methodology chapter gives a detailed systematic approach aiming to discover new natural products from phytopathogenic fungi and characterize their role in triggering plant cell death and plant disease. This approach takes advantage of the global regulation of fungal secondary metabolite production by regulatory proteins reported in various fungal species.

Recent advances in the biosynthesis of ribosomally synthesized and posttranslationally modified peptides of fungal origin.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are growing class of natural products with potent biological activities. Although the core scaffolds of RiPPs are composed of proteinogenic amino acids, remarkable structural diversity is generated through posttranslational modifications (PTMs) of precursor peptides. In addition, ribosomal origin of biosynthetic precursors enables supply of its analogs through genetic approach such as site-directed mutagenesis on corresponding genes. As PTM enzymes often exhibit substrate tolerance, RiPP biosynthetic machineries are considered as efficient tools for generation of unique peptide derivatives. RiPP pathways are distributed among all domains of life and those derived from bacteria and plants have been known for decades. In contrast, fungal RiPPs (F-RiPPs) have fewer examples. Amatoxins and omphalotins are F-RiPPs produced by Basidiomycota fungi. In the biosynthesis of these compounds, macrocyclization by prolyl oligopeptidase homologs and N-methylations of back bone amides have been characterized, respectively. Ustiloxins and related compounds are another group of F-RiPPs with characteristic macrocyclic ethers. UstYa family proteins, which are fungi-specific putative oxidases, have been identified as common proteins involved in PTMs of these compounds. Despite a limited number of characterized examples, recent progress in sequencing of fungal genomes indicated that a number of RiPP pathways are hidden in fungal resources, making F-RiPPs as attractive target for genome mining studies while more detailed understandings of key biosynthetic enzymes are still necessary. This review seeks to describe recent advances on the F-RiPP biosynthesis with slight emphasis on the function of UstYa family proteins.

An Improved Genome Sequence Resource of Bipolaris maydis, Causal Agent of Southern Corn Leaf Blight.

An Improved Genome Sequence Resource of Bipolaris maydis, Causal Agent of Southern Corn Leaf Blight.

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

Rhizophagus proliferus genome sequence reiterates conservation of genetic traits in AM fungi, but predicts higher saprotrophic activity.

Arbuscular mycorrhizal (AM) fungi are ubiquitous endosymbionts of terrestrial plants. It helps plants to extract more nutrients from the soil and enhances the plant tolerance to various ecological stress factors. The AM fungal genome sequence helps to identify the gene repertoires that are crucial for adaptation to different habitat and mechanisms for interaction with host plant. The present work comprises the first draft of the genome sequence of Rhizophagus proliferus, which is an important AM species present in biofertilizer consortia for agricultural purpose. The estimated genome size of R. proliferus is ~ 110 Mbps and its genomic assembly is 94.35% complete. Genome mining was carried out to identify putative gene families important for biological functions. A total of 22,526 protein-coding genes were estimated in the genome, with an abundance of kinases and reduced number of glycoside hydrolases as compared to other fungal classes. A striking finding in the R. proliferus genome was higher number of carbohydrate esterases (CE), which may suggest towards presence of higher saprotrophic activity in this species as compared to the previously reported AM fungi, which may indicate towards its role as a link between plants and soil mineral nutrients. The genome sequence and annotation of R. proliferus presented here would serve as an important reference for functional genomics studies required for developing biofertilizer formulations in future. In addition, the findings from this work may also prove important in deciphering molecular mechanisms in AM fungi that govern the host-specific interaction and associated agriculture benefits.

Difficulty in Assigning Fungal Identity Based on DNA Sequences.

The Comment Letter by Houbraken et al. (1) correctly points out the need for accurate fungal taxonomical identification in biological research and the difficulty confronted by scientists in assigning genus and species identity due to the high genome sequence similarity of related individuals and the low availability of full fungal genome sequences in DNA data repositories with which to perform sequence comparisons. The Comment Letter accurately presents the need to revise or even update previously assigned fungal classifications as more DNA sequences and full genomes become available over time. The Comment Letter highlights that currently, the internal transcribed spacer (ITS) region is most often used to identify fungi, and the authors argue that some short regions of genes such as benA, caM, and tef1-alpha may be better suited for accurate species identification of close relatives. Based on that premise, the authors used the partial beta-tubulin (benA) and partial calmodulin (caM) gene sequences to reassess the taxonomical identity of multiple fungal isolates published in journals such as MRA (previously known as Genome Announcements) using phylogenetic trees. The authors argue that upon analysis of the short regions of the benA or caM genes, 21 published papers in MRA had incorrectly assigned the fungal identity of their isolates. While we agree that assigned fungal taxa should be revised as more DNA sequence information becomes available, this does not mean that the classification provided at the time of publication was erroneous based on the available sequence information at the time. Furthermore, we argue that the authors actually contradicted their own argument when they ignored available ITS sequences, 18S rRNA gene sequences, and even full genome sequences and relied on a very short 65-amino acid region of the benA gene to assign fungal identity. Based on the 65-amino acid region of benA, Houbraken et al. (1) claimed that our paper (2) misidentified the isolate as Byssochlamys sp., when it should have been Monascus floridanus.

Widespread bacterial diversity within the bacteriome of fungi

Knowledge of associations between fungal hosts and their bacterial associates has steadily grown in recent years as the number and diversity of examinations have increased, but current knowledge is predominantly limited to a small number of fungal taxa and bacterial partners. Here, we screened for potential bacterial associates in over 700 phylogenetically diverse fungal isolates, representing 366 genera, or a tenfold increase compared with previously examined fungal genera, including isolates from several previously unexplored phyla. Both a 16 S rDNA-based exploration of fungal isolates from four distinct culture collections spanning North America, South America and Europe, and a bioinformatic screen for bacterial-specific sequences within fungal genome sequencing projects, revealed that a surprisingly diverse array of bacterial associates are frequently found in otherwise axenic fungal cultures. We demonstrate that bacterial associations with diverse fungal hosts appear to be the rule, rather than the exception, and deserve increased consideration in microbiome studies and in examinations of microbial interactions.

New Method for Identifying Fungal Kingdom Enzyme Hotspots from Genome Sequences.

Fungal genome sequencing data represent an enormous pool of information for enzyme discovery. Here, we report a new approach to identify and quantitatively compare biomass-degrading capacity and diversity of fungal genomes via integrated function-family annotation of carbohydrate-active enzymes (CAZymes) encoded by the genomes. Based on analyses of 1932 fungal genomes the most potent hotspots of fungal biomass processing CAZymes are identified and ranked according to substrate degradation capacity. The analysis is achieved by a new bioinformatics approach, Conserved Unique Peptide Patterns (CUPP), providing for CAZyme-family annotation and robust prediction of molecular function followed by conversion of the CUPP output to lists of integrated “Function;Family” (e.g., EC 3.2.1.4;GH5) enzyme observations. An EC-function found in several protein families counts as different observations. Summing up such observations allows for ranking of all analyzed genome sequenced fungal species according to richness in CAZyme function diversity and degrading capacity. Identifying fungal CAZyme hotspots provides for identification of fungal species richest in cellulolytic, xylanolytic, pectinolytic, and lignin modifying enzymes. The fungal enzyme hotspots are found in fungi having very different lifestyle, ecology, physiology and substrate/host affinity. Surprisingly, most CAZyme hotspots are found in enzymatically understudied and unexploited species. In contrast, the most well-known fungal enzyme producers, from where many industrially exploited enzymes are derived, are ranking unexpectedly low. The results contribute to elucidating the evolution of fungal substrate-digestive CAZyme profiles, ecophysiology, and habitat adaptations, and expand the knowledge base for novel and improved biomass resource utilization.

Identification, heterologous production and bioactivity of lentinulin A and dendrothelin A, two natural variants of backbone N-methylated peptide macrocycle omphalotin A

Backbone N-methylation and macrocyclization improve the pharmacological properties of peptides by enhancing their proteolytic stability, membrane permeability and target selectivity. Borosins are backbone N-methylated peptide macrocycles derived from a precursor protein which contains a peptide α-N-methyltransferase domain autocatalytically modifying the core peptide located at its C-terminus. Founding members of borosins are the omphalotins from the mushroom Omphalotus olearius (omphalotins A-I) with nine out of 12 L-amino acids being backbone N-methylated. The omphalotin biosynthetic gene cluster codes for the precursor protein OphMA, the protease prolyloligopeptidase OphP and other proteins that are likely to be involved in other post-translational modifications of the peptide. Mining of available fungal genome sequences revealed the existence of highly homologous gene clusters in the basidiomycetes Lentinula edodes and Dendrothele bispora. The respective borosins, referred to as lentinulins and dendrothelins are naturally produced by L. edodes and D. bispora as shown by analysis of respective mycelial extracts. We produced all three homologous peptide natural products by coexpression of OphMA hybrid proteins and OphP in the yeast Pichia pastoris. The recombinant peptides differ in their nematotoxic activity against the plant pathogen Meloidogyne incognita. Our findings pave the way for the production of borosin peptide natural products and their potential application as novel biopharmaceuticals and biopesticides.

A CRISPR/Cas9 Cleavage System for Capturing Fungal Secondary Metabolite Gene Clusters.

More and more available fungal genome sequence data reveal a large amount of secondary metabolite (SM) biosynthetic 'dark matter' to be discovered. Heterogeneous expression is one of the most effective approaches to exploit these novel natural products, but it is limited by having to clone entire biosynthetic gene clusters (BGCs) without errors. So far, few effective technologies have been developed to manipulate the specific large DNA fragments in filamentous fungi. Here, we developed a fungal BGC-capturing system based on CRISPR/Cas9 cleavage in vitro. In our system, Cas9 protein was purified and CRISPR guide sequences in combination with in vivo yeast assembly were rationally designed. Using targeted cleavages of plasmid DNAs with linear (8.5 kb) or circular (8.5 kb and 28 kb) states, we were able to cleave the plasmids precisely, demonstrating the high efficiency of this system. Furthermore, we successfully captured the entire Nrc gene cluster from the genomic DNA of Neosartorya fischeri. Our results provide an easy and efficient approach to manipulate fungal genomic DNA based on the in vitro application of Cas9 endonuclease. Our methodology will lay a foundation for capturing entire groups of BGCs in filamentous fungi and accelerate fungal SMs mining.

Molecular identification of Cryptococcus gattii from cerebrospinal fluid using single-cell sequencing: A case study

A 31-year-old man presented with cryptococcal meningitis (CM) without typical clinical characteristics, but with abnormal walking, difficult leg lifting and frequent falling. He was admitted to Peking Union Medical College Hospital. After multiple tests failed to identify the pathogen, single-cell sequencing (scS) was used to test the cerebrospinal fluid (CSF). Comparing the sequence obtained from single-cell sequencing with the reference database, it was found that the infection was caused by Cryptococcus gattii sensu stricto (AFLP4/VGI genotype). Cryptococcus is difficult to cultivate from complex body fluids. The etiological agent of this patient was identified and the patient was treated. This is the first case in which scS was used to detect and identify fungal pathogen after conventional testing failed to identify the cause of the disease. This report demonstrates that the scS approach can be used to generate fungal genome sequences directly from the CSF of a CM patient. The scS technology could become a powerful tool to precise detect microscopically visible but uncultured pathogens in clinical samples.

Recurrent Loss of abaA, a Master Regulator of Asexual Development in Filamentous Fungi, Correlates with Changes in Genomic and Morphological Traits.

Gene regulatory networks (GRNs) drive developmental and cellular differentiation, and variation in their architectures gives rise to morphological diversity. Pioneering studies in Aspergillus fungi, coupled with subsequent work in other filamentous fungi, have shown that the GRN governed by the BrlA, AbaA, and WetA proteins controls the development of the asexual fruiting body or conidiophore. A specific aspect of conidiophore development is the production of phialides, conidiophore structures that are under the developmental control of AbaA and function to repetitively generate spores. Fungal genome sequencing has revealed that some filamentous fungi lack abaA, and also produce asexual structures that lack phialides, raising the hypothesis that abaA loss is functionally linked to diversity in asexual fruiting body morphology. To examine this hypothesis, we carried out an extensive search for the abaA gene across 241 genomes of species from the fungal subphylum Pezizomycotina. We found that abaA was independently lost in four lineages of Eurotiomycetes, including from all sequenced species within the order Onygenales, and that all four lineages that have lost abaA also lack the ability to form phialides. Genetic restoration of abaA from Aspergillus nidulans into Histoplasma capsulatum, a pathogenic species from the order Onygenales that lacks an endogenous copy of abaA, did not alter Histoplasma conidiation morphology but resulted in a marked increase in spore viability. We also discovered that species lacking abaA contain fewer AbaA binding motifs in the regulatory regions of orthologs of some AbaA target genes, suggesting that the asexual fruiting body GRN of organisms that have lost abaA has likely been rewired. Our results provide an illustration of how repeated losses of a key regulatory transcription factor have contributed to the diversity of an iconic fungal morphological trait.

Chemical, Bioactivity, and Biosynthetic Screening of Epiphytic Fungus Zasmidium pseudotsugae.

We report the first secondary metabolite, 8,8′-bijuglone, obtained from pure cultures of the slow growing Douglas fir- (Pseudotsuga menziesii var. menziesii) foliage-associated fungus Zasmidium pseudotsugae. The quinone was characterized using extensive LC/MS and NMR-based spectroscopic methods. 8,8′-Bijuglone exhibited moderate antibiotic activity against Gram-positive pathogens and weak cytotoxic activity in the NCI-60 cell line panel and in our in-house human colon carcinoma (HCT-116) cell line. An analysis of the fungal genome sequence to assess its metabolic potential was implemented using the bioinformatic tool antiSMASH. In total, 36 putative biosynthetic gene clusters were found with a majority encoding for polyketides (17), followed by non-ribosomal peptides (14), terpenes (2), ribosomal peptides (1), and compounds with mixed biosynthetic origin (2). This study demonstrates that foliage associated fungi of conifers produce antimicrobial metabolites and suggests this guild of fungi may present a rich source of novel molecules.

Animal, Fungi, and Plant Genome Sequences Harbor Different Non-Canonical Splice Sites.

Most protein-encoding genes in eukaryotes contain introns, which are interwoven with exons. Introns need to be removed from initial transcripts in order to generate the final messenger RNA (mRNA), which can be translated into an amino acid sequence. Precise excision of introns by the spliceosome requires conserved dinucleotides, which mark the splice sites. However, there are variations of the highly conserved combination of GT at the 5′ end and AG at the 3′ end of an intron in the genome. GC-AG and AT-AC are two major non-canonical splice site combinations, which have been known for years. Recently, various minor non-canonical splice site combinations were detected with numerous dinucleotide permutations. Here, we expand systematic investigations of non-canonical splice site combinations in plants across eukaryotes by analyzing fungal and animal genome sequences. Comparisons of splice site combinations between these three kingdoms revealed several differences, such as an apparently increased CT-AC frequency in fungal genome sequences. Canonical GT-AG splice site combinations in antisense transcripts are a likely explanation for this observation, thus indicating annotation errors. In addition, high numbers of GA-AG splice site combinations were observed in Eurytemora affinis and Oikopleura dioica. A variant in one U1 small nuclear RNA (snRNA) isoform might allow the recognition of GA as a 5′ splice site. In depth investigation of splice site usage based on RNA-Seq read mappings indicates a generally higher flexibility of the 3′ splice site compared to the 5′ splice site across animals, fungi, and plants.